Heat shock or stress response is a phenomenon observed in living cells of all types that have been exposed at least temporarily to a temperature a few degrees above physiological growth temperature. One manifestation of this response is the appearance within the cells of abnormally folded proteins in general. Another manifestation is the increased expression of a family of proteins which under normal growth conditions are expressed by the same cells at lower levels. These proteins have therefore been termed "heat shock proteins" or, more recently, "stress proteins." The increased expression of the stress proteins and the accumulation of abnormally folded forms of other proteins have also been observed in cells exposed to a variety of metals, amino acids, ethanol and other conditions and treatments.
Another manifestation of the response is the development or acquisition of a thermotolerant phenotype. These phenotypes are developed by subjecting cells and cell masses such as tissues and organs to mild heat shock and then allowing them to recover at normal growth temperature. The thermotolerance thus acquired enables these cells to more effectively withstand a subsequent and more severe heat shock treatment which would otherwise do irreversible damage to the cells. Thermotolerant phenotypes also occur in cells exposed to other agents or treatments which elicit stress responses, such as heavy metals, arsenite, various amino acid analogs, and other metabolic poisons such as the sulfhydryl reducing agents iodoacetamide and p-chloromercuribenzoate. Thus, the stress response elicited by one particular agent or treatment can render the cells tolerant upon their exposure to a different agent or treatment which also can result in increased synthesis of one or more of the stress proteins. This is known as "cross-protection." A still further characteristic of thermotolerant phenotypes is "translational thermotolerance," which relates to both the rate of protein synthesis in general, the extent of protein synthesis, or both, by a cell after exposure to heat shock. In normal cells (those not yet made thermotolerant), protein synthesis rates drop upon exposure to heat shock and require considerable time to return to normal. In thermotolerant phenotypes, the recovery of protein synthesis is considerably faster.
Thermotolerance phenotypes have been induced in vivo in intact organisms and also in organs and tissues. Although the bulk of the experiments utilize hyperthermic treatments to induce thermotolerance, a growing body of data demonstrates that thermotolerance can be induced by treatment with certain chemical agents. For example, heat shock proteins have been induced in various cell cultures by treating them with agents such as sodium arsenite, cadmium chloride, cycloheximide, steroids, ethanol and nitric oxide.
Chemical agents have been used to induce the synthesis of heat shock proteins and for converting organisms and organs to a thermotolerant phenotype. For example, the insect hormone .alpha.-ecdysterone, induces thermotolerance and the synthesis of heat shock proteins in Drosophila (Buzin et al., "The induction of a subset of heat shock proteins by drugs that inhibit differentiation in Drosophila embryonic cell cultures;" In, HEAT SHOCK: FROM BACTERIA TO MAN, Schlesinger et al., Eds. Cold Spring Harbor, New York, pp. 387-394 (1982)). In Drosophila, an increased thermotolerance arises concomitant with the synthesis of heat shock proteins (Berger et al., Small heat shock proteins in Drosophila may confer thermal tolerance, Exp. Cell. Res. 147:437-442 (1983)). In rats, heat shock protein synthesis and thermotolerance have been induced by treating the experimental animals with sodium arsenite. Sodium arsenite induced heat shock synthesis in the rats, particularly in the lungs. The heat shock protein (hsp 72) was detected as early as 2 hours following arsenite injection. The expression of heat shock proteins correlated with significant protection against cecal ligation and perforation induced mortality (Ribeiro et al., "Sodium arsenite induces heat shock protein-72 kilodalton expression in the lungs and protects rats against sepsis," Critical Care Medicine 22:922-929 (1994)).
While it is logical to speculate that the increased expression of the stress proteins is in some way related to the acquisition of thermotolerance, the actual basis by which the tolerant phenotype is manifested is still unclear. For example, the proteins whose rate of expression is increased by heat shock range widely in molecular weight, some being in the 20,000 dalton range and others ranging as high as 110,000 daltons, and the same proteins are not always increased at the same rates in all species. In addition to the stress proteins, thermotolerance is accompanied by other physiological changes which result from the initial priming stress treatment. These include activation of protein kinase/phosphatase cascades, rearrangements of the cytoskeleton, membrane fluidity changes, changes in intracellular ions, and changes in cell growth and cell cycle. The type of contribution made by the stress proteins and the degree and manner in which these proteins interact with these other physiological changes raise many questions about the mechanism by which thermotolerance is actually achieved. Although there is broad correlation in the literature between heat shock protein synthesis and the induction of thermotolerance, in view of the multitude of factors involved, one cannot definitively conclude that an observed increase in the expression of certain stress proteins is a clear indication that thermotolerance will follow.
Further background information relevant to this invention is found in reports on the class of antibiotics known as benzoquinonoid ansamycins. These include the herbimycins A, B and C, geldanamycin, and various derivatives and analogs of these compounds. These compounds are known to exhibit antitumor activity and, in the case of the herbimycins, herbicidal, antiviral and anti-angiogenic activity as well. Explorations of the antitumor activity of these compounds have shown that these compounds inhibit p60.sup.v-src, a tyrosine-specific protein kinase, and thereby reverse the transformation of Rous sarcoma virus-transformed cells, possibly by binding to the kinase. More recent studies have suggested that these compounds can bind to hsp90. Whiteseil, L., et al., "Inhibition of heat shock protein HSP90-pp60.sup.v-src heteroprotein complex formation by benzoquinone ansamycins: Essential role for stress proteins in oncogenic transformation," Proc. Natl. Acad. Sci. USA 91:8324-8328 (1994). Because hsp90 is known to be important for the maturation of p60.sup.v-src, reversion of the transformed phenotype may be due to the inability of the cells in the presence of the benzoquinonoid ansamycins to properly produce active and mature p60.sup.v-src.
In the course of these explorations, it was discovered that herbimycin A induced the synthesis of a 70-kDa protein in A431 human epidermoid carcinoma cells, and that this protein is one of the heat stress proteins referred to above. Murakami, Y., et al., "Induction of hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes," Experimental Cell Research 195:338-344 (1991). In their conclusions from these findings, however, Murakami, et al. acknowledged that although the heat shock proteins are thought to play a role in certain cellular processes, the exact function of heat shock proteins remains obscure. It is significant to note that Murakami, et al. speculate that herbimycin A may associate with newly synthesized proteins, in particular the EGF receptors, and thereby inhibit proper maturation of the receptors. In addition, they suggested that this interference with EGF receptor maturation might lead to increased hsp70 synthesis. Murakami, et al. refrain from any reference to thermotolerance, or from speculating as to whether herbimycin A itself can create thermotolerance, aside from its ability to induce the one stress protein that the authors observed. Murakami, et al. also suggested that other proteins (60- and 90-kilodalton) were increased as a result of herbimycin A exposure, but the authors did not prove that these proteins were heat shock proteins.
Accordingly, given the knowledge of the complexity of the heat shock response, there is no suggestion in the literature that thermotolerance can be induced by any means other than agents or treatments which lead to the general accumulation of abnormally folded proteins.